Synthesis of Nuxia oppositifolia nanoparticles

ABSTRACT

Synthesis of  Nuxia oppositifolia  nanoparticles includes providing an extract of  Nuxia oppositifolia , dissolving the extract in alcohol to provide a mixture, adding water to the mixture to provide an aqueous solution, adding an acidic solution to the aqueous solution to form a final solution including  Nuxia oppositifolia  nanoparticles; and centrifuging the final solution to isolate the  Nuxia oppositifolia  nanoparticles.

BACKGROUND OF THE INVENTION 1. Field of the Invention

The present invention relates to nanoparticle synthesis, and particularly to a synthesis of Nuxia oppositifolia nanoparticles.

2. Description of the Related Art

Nanoparticles exhibit completely new properties compared to their corresponding bulk materials. Nanoparticles have found tremendous application in the fields of high sensitivity bimolecular detection and diagnostics, therapeutics, antimicrobials, and nanomedicine.

The genus Nuxia (family Buddlejaceae) includes about fifteen species of shrubs and trees spread over the Arabian peninsula, tropical and South Africa. It has been found that the dichloromethane leaf extract of N. verticillata, endemic to the Mascarene Archipelago, has potential anti-proliferative activity (IC₅₀=44 ug/ml) and selectively inhibits cancer cell growth in comparison with normal cells.

Nuxia is represented in Saudi Arabia by two species, N. oppositifolia and N. congesta, which grow in the forests below the juniper zone, usually in riverine habitats. Many Nuxia species are plants of economic and medicinal interest with a rich diversity of ethnobotanical uses. Leaves of N. floribunda are reported to be used for infantile convulsions in parts of Africa. Nuxia oppositifolia (Hochst.) Benth., known as Water elder, is an ever green shrub. In the traditional medicine of southern Africa, the smoke of the burning leaves of Nuxia oppositifolia is inhaled to treat headache. In Madagascar, Nuxia oppositifolia leaf decoction is used to treat malaria in children.

SUMMARY OF THE INVENTION

Synthesis of Nuxia oppositifolia nanoparticles includes providing an extract of Nuxia oppositifolia, dissolving the extract in alcohol to provide a mixture, adding water to the mixture to provide an aqueous solution, adding an acidic solution to the aqueous solution to form a final solution including Nuxia oppositifolia nanoparticles; and centrifuging the final solution to isolate the Nuxia oppositifolia nanoparticles.

These and other features of the present invention will become readily apparent upon further review of the following specification and drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing the particles size distribution of Nuxia oppositifolia nanoparticles synthesized according to the present invention.

FIGS. 2A-2B are TEM images of the Nuxia oppositifolia nanoparticles synthesized according to the present invention.

FIG. 3 is a graph showing the cytotoxicity of the Nuxia oppositifolia nanoparticles synthesized according to the present invention against HeLa cell line.

FIG. 4 is a graph showing the cytotoxicity of the Nuxia oppositifolia nanoparticles synthesized according to the present invention against A-549 cell line.

FIG. 5 is a graph showing the cytotoxicity of the Nuxia oppositifolia nanoparticles synthesized according to the present invention against MCF7 cell line.

FIG. 6 is a graph showing the cytotoxicity of the Nuxia oppositifolia nanoparticles synthesized according to the present invention against HepG-2 cell line.

FIG. 7 is a graph showing the cytotoxicity of the Nuxia oppositifolia nanoparticles synthesized according to the present invention against HCT-116 cell line.

Similar reference characters denote corresponding features consistently throughout the attached drawings.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Synthesis of Nuxia oppositifolia nanoparticles includes providing an extract of Nuxia oppositifolia, dissolving the extract in alcohol to provide a mixture, adding water to the mixture to provide an aqueous solution, adding an acidic solution to the aqueous solution to form a final solution including Nuxia oppositifolia nanoparticles; and centrifuging the final solution to isolate the Nuxia oppositifolia nanoparticles. The extract can be a dried extract. The alcohol can be methanol. The acidic solution can be hydrochloric acid solution, e.g., 38% hydrochloric acid solution. The acidic solution can be added dropwise to the aqueous solution while stirring at a temperature of about 30±2° C. The final solution can be centrifuged at about 9000 rpm for about fifteen minutes.

The dried extract of Nuxia oppositifolia can be prepared by drying aerial parts, e.g., leaves and stems, of the Nuxia oppositifolia plant, and macerating with alcohol, e.g., ethanol, at room temperature. The resulting alcohol extract of Nuxia oppositifolia can be filtered and concentrated under reduced pressure, e.g., at 40° C. using a rotary evaporator, to provide a dried extract of Nuxia oppositifolia.

The Nuxia oppositifolia nanoparticles can be used to effectively treat cancer. An effective amount of the Nuxia oppositifolia nanoparticles can be administered to a patient with cancer. The Nuxia oppositifolia nanoparticles can be used in combination with one or more additional treating agents for cancer.

The following examples illustrate the present teachings.

Example 1 Synthesis of Nuxia oppositifolia Nanoparticles

The fresh aerial parts (leaves and stems) of Nuxia oppositifolia were collected from Wadi Lajab in Jizan, located in the south part of Saudi Arabia. The plant was deposited at the Herbarium of the College of Pharmacy, King Saud University, Riyadh, Saudi Arabia.

The air-dried and powdered aerial parts of N. oppositifolia (500 g) were extracted by maceration with 70% ethanol (5×1 L) at room temperature. The combined obtained ethanolic extract was filtered and concentrated under reduced pressure at 40° C. using a rotary evaporator, to give a dried ethanolic extract (35 g).

Three grams of N. oppositifolia dried ethanolic extract were dissolved in 50 mL methanol under stirring, then 5 ml distilled water was added. After that a specific amount of 38% hydrochloric acid solution was added drop wise. The flask was kept under stirring at a temperature of 30±2° C. The N. oppositifolia nanoparticles were centrifuged (9000 rpm) for 15 minutes to collect the nanoparticles.

The synthesized nanoparticles were characterized using Zetasizer, Nano series, HT Laser, ZEN3600 from Molvern Instrument, UK to determine the average size of the resulting nanoparticles. Transmission electron microscopy (TEM, JEM-1400, JEOL, Japan) was employed to characterize the size, shape and morphologies of nanoparticles. FIG. 1 shows the particle size distribution of the Nuxia oppositifolia nanoparticles, with the Z-average particle size being 287 nm. FIGS. 2A-2B show TEM images of the Nuxia oppositifolia nanoparticles.

Example 2 Cytotoxicity Evaluation of Nuxia oppositifolia Nanoparticles

Dimethyl sulfoxide (DMSO), crystal violet and trypan blue dye were purchased from Sigma (St. Louis, Mo., USA). Fetal Bovine serum, DMEM, RPMI-1640, HEPES buffer solution, L-glutamine, gentamycin and 0.25% Trypsin-EDTA were purchased from (Bio Whittaker® Lonza, Belgium).

Crystal violet, composed of 0.5% (w/v) crystal violet and 50% methanol, was used as staining solution. The mammalian cell lines for HepG2 cells (human cell line of a well differentiated hepatocellular carcinoma isolated from a liver biopsy of a male Caucasian aged 15 years) were obtained from the American Type Culture Collection (ATCC), while the MCF-7 cells (human breast cancer cell line) were obtained from VACSERA Tissue Culture Unit.

The cells were propagated in Dulbeccos modified Eagles Medium (DMEM) supplemented with 10% heat inactivated fetal bovine serum, 1% L-glutamine, HEPES buffer and 50 μg/mL gentamicin. Cells were maintained at 37° C. in a humidified atmosphere with 5% CO2 and were sub-cultured two times a week.

The cytotoxic activity was evaluated by the crystal violet staining (CVS) method. Saotome K., Morita, H., and Umeda, M. (1989). “Cytotoxicity test with simplified crystal violet staining method using microtitre plates and its application to injection drugs.” Toxicol. In Vitro, 3: 317-321.

Briefly, the cells were seeded in a 96-well tissue culture microplate, at a concentration of 1×104 cells per well in 100 μL of growth medium at 37° C. After 24 hours of seeding, fresh medium containing various concentrations of the tested compounds (50 μg, 25 μg, 12.5 μg, 6.25 μg, 3.125 μg & 1.56 μg) were added to the microtiter plates (each compound was tested in triplicate in all concentrations). Next, the microtiter plates were incubated at 37° C. in a humidified incubator with 5% CO₂. Control cells were incubated without test sample and with or without DMSO. The little percentage of DMSO present in the wells was found not to affect the experiment. After 48 hours incubation period, viable cells yield was determined by a colorimetric method.

In brief, media were aspirated for 30 min and the crystal violet solution (1%) was added to each well. The plates were rinsed after removing the stain by distilled water. Glacial acetic acid (30%) was then added to all wells and mixed thoroughly. The quantitative analysis, to evaluate the fixed cells, was performed calorimetrically by measuring the absorbance in an automatic Microplate reader (TECAN, Inc.) at 595 nm. The effect on cell growth was calculated as the difference in absorbance percentage in the presence and absence of the tested compounds. As discussed further below, a dose-response curve was plotted to acquire the concentration at which the growth of cells was inhibited to 50% of the control (IC₅₀). The standard antitumor drug used was doxorubicin.

The cytotoxic effects of the synthesized nanoparticles were evaluated against the five cancer cell lines, namely the cervical carcinoma cells HELA (FIG. 3, Table 1), the human lung adenocarcinoma epithelial cell line A549 (FIG. 4, Table 2), the breast carcinoma cells MCF-7 (FIG. 5, Table 3), the hepatocellular carcinoma cells HepG-2 (FIG. 6, Table 4) and the human colon carcinoma cells HCT-116 (FIG. 7, Table 5). The results, provided in Tables 1-5 and FIGS. 3-7, indicate that the N. oppositifolia nanoparticles are effective in inhibiting cancer cells.

TABLE 1 Evaluation of cytotoxicity of synthesized N. oppositifolia nanoparticles against HeLa cell line with IC₅₀ = 39.7 μl/ml. Sample conc. % Viability (3 Replicates) % Standard (μl/ml) 1^(st) 2^(nd) 3^(rd) Mean Inhibition Deviation (±) 100 24.87 18.68 21.85 21.80 78.20 3.10 50 41.92 38.74 40.72 40.46 59.54 1.61 25 63.84 64.89 61.95 63.56 36.44 1.49 12.5 79.68 78.91 74.18 77.59 22.41 2.98 6.25 86.19 89.47 88.96 88.21 11.79 1.77 3.125 94.02 97.24 95.17 95.48  4.52 1.63

TABLE 2 Evaluation of cytotoxicity of synthesized N. oppositifolia nanoparticles against A-549 cell line with IC₅₀ = 11.2 μl/ml. Sample conc. % Viability (3 Replicates) % Standard (μl/ml) 1^(st) 2^(nd) 3^(rd) Mean Inhibition Deviation (±) 100  7.38 10.12  9.58  9.03 90.97 1.45 50 18.04 19.73 16.46 18.08 81.92 1.64 25 32.56 34.64 30.83 32.68 67.32 1.91 12.5 43.25 46.51 41.92 43.89 56.11 2.36 6.25 78.72 69.65 73.41 73.93 26.07 4.56 3.125 90.21 85.18 87.06 87.48 12.52 2.54

TABLE 3 Evaluation of cytotoxicity of synthesized N. oppositifolia nanoparticles against MCF7 cell line with IC₅₀ = 19.2 μl/ml. Sample conc. % Viability (3 Replicates) % Standard (μl/ml) 1^(st) 2^(nd) 3^(rd) Mean Inhibition Deviation (±) 100 16.62 19.73 20.45 18.93 81.07 2.04 50 30.47 34.06 33.91 32.81 67.19 2.03 25 41.29 43.97 45.82 43.69 56.31 2.28 12.5 57.12 60.49 54.23 57.28 42.72 3.13 6.25 65.81 74.25 68.19 69.42 30.58 4.35 3.125 78.49 85.14 81.36 81.66 18.34 3.34

TABLE 4 Evaluation of cytotoxicity of synthesized N. oppositifolia nanoparticles against HepG-2 cell line with IC₅₀ ₌ 21.9 μl/ml. Sample conc. % Viability (3 Replicates) % Standard (μl/ml) 1^(st) 2^(nd) 3^(rd) Mean Inhibition Deviation (±) 100 21.38 18.79 20.61 20.26 79.74 1.33 50 34.62 36.28 32.75 34.55 65.45 1.77 25 47.95 45.12 43.86 45.64 54.36 2.09 12.5 65.18 63.57 60.24 63.00 37.00 2.52 6.25 78.64 76.38 81.49 78.84 21.16 2.56 3.125 89.76 85.93 90.62 88.77 11.23 2.50

TABLE 5 Evaluation of cytotoxicity of synthesized N. oppositifolia nanoparticles against HCT-116 cells was detected with IC₅₀ = 10.9 μl/ml. Sample Standard conc. % Viability (3 Replicates) % Deviation (μl/ml) 1^(st) 2^(nd) 3^(rd) Mean Inhibition (±) 100 12.98 8.73 9.65 10.45 89.55 2.24 50 20.64 21.35 18.43 20.14 79.86 1.52 25 32.87 35.69 31.98 33.51 66.49 1.94 12.5 42.19 40.83 45.06 42.69 57.31 2.16 6.25 69.51 67.08 74.87 70.49 29.51 3.99 3.125 87.24 85.43 90.65 87.77 12.23 2.65 0 100 100 100 100  0.00

Example 3 Antimicrobial Activity Evaluation of Nuxia oppositifolia Nanoparticles

The antimicrobial effect of N. oppositifolia nanoparticles were evaluated against gram positive bacteria, gram negative bacteria, as well as fungi. Results of the antimicrobial effect of the nanoparticles are provided in Table 6.

TABLE 6 Antimicrobial activity of synthesized N. oppositifolia nanoparticles using agar diffusion technique. Sample Zone of Inhibition Tested microorganisms (±SD) Reference Drug Fungi B Amphotericin Absidi corymbifera 21.3 ± 0.22 23.0 ± 0.10 (RCMB 02564) Geotricum candidum 23.0 ± 0.14 27.0 ± 0.20 (RCMB 05097) Candida albicans 20.3 ± 0.46 25.7 ± 0.10 (RCMB 05036) Gram Positive bacteria: Ampicillin Staphylococcus aureus 24.0 ± 0.42 27.3 ± 0.14 (RCMB 010027) Staphylococcus epidermidis 25.3 ± 0.23 25.0 ± 0.18 (RCMB 010024) Streptococcus pyogenes 23.6 ± 0.89 26.3 ± 0.34 (RCMB 010015) Gram Negative bacteria: Gentamycin Proteous vulgaris 23.6 ± 0.63 23.4 ± 0.30 (RCMB 010085) Klebsiella pneumoniae 25.3 ± 0.12 26.4 ± 0.15 (RCMB 0010093) Salmonella enteritidis 25.3 ± 0.68 25.2 ± 0.18 (RCMB 010084) Data are expressed in the form of mean ±S.D. beyond well diameter (6 mm); 100 ul of samples were tested.

It is to be understood that the present invention is not limited to the embodiments described above, but encompasses any and all embodiments within the scope of the following claims. 

We claim:
 1. A method of synthesizing Nuxia oppositifolia nanoparticles, comprising: providing an extract of Nuxia oppositifolia; dissolving the extract in alcohol to provide a mixture; adding water to the mixture to provide an aqueous solution; adding an effective amount of an acidic solution to the aqueous solution to form a final solution containing Nuxia oppositifolia nanoparticles; and centrifuging the final solution at 9000 rpm for 15 minutes to isolate and collect the Nuxia oppositifolia nanoparticles.
 2. The method of synthesizing Nuxia oppositifolia nanoparticles according to claim 1, wherein the alcohol is methanol.
 3. The method of synthesizing Nuxia oppositifolia nanoparticles according to claim 1, wherein the acidic solution is hydrochloric acid solution.
 4. The method of synthesizing Nuxia oppositifolia nanoparticles according to claim 1, wherein the nanoparticles have an average particle size of about 287 nm.
 5. The method of synthesizing Nuxia oppositifolia nanoparticles according to claim 1, wherein the extract is a dried extract.
 6. The method of synthesizing Nuxia oppositifolia nanoparticles according to claim 5, wherein the dried extract is prepared by: drying aerial parts of Nuxia oppositifolia; macerating the dried aerial parts with alcohol to provide an alcohol extract; filtering the alcohol extract; and drying the filtered alcohol extract under reduced pressure to provide the dried extract.
 7. The method of synthesizing Nuxia oppositifolia nanoparticles according to claim 6, wherein the alcohol is ethanol. 